Project Summary Filoviruses, which include the ebolaviruses and marburgviruses, are non-segmented, negative-sense RNA viruses (NNSVs) that cause severe human disease. These viruses are of concern as emerging pathogens and as potential bioterrorism threats. Their importance and public health impact are reinforced by the West Africa epidemic that began in winter of 2014 and has resulted in more than 11,000 deaths and the export of Ebola virus disease to the U.S., the U.K. and Europe. Although the past year has seen progress toward development of effective vaccines and treatments, current prophylactic and treatment options remain limited. Particularly lacking are effective small molecule inhibitors. Complicating development of anti-filovirus drugs is the biosafety level 4 (BSL4) containment needed to work with live filoviruses, which is only available at a few locations worldwide. With substantial restrictions on the number of investigators who have access to such facilities, antiviral testing against infectious virus in a high throughput setting is problematic. An alternate approach is to develop assays of specific viral functions that can be assessed without generation of infectious materials. The viral RNA-dependent RNA polymerase (RDRP) complex is a particularly promising candidate. The complex consists of the viral nucleoprotein (NP), viral protein of 35KDa (VP35), VP30 and the large protein (L) which is the enzymatic component of the complex and the only enzyme encoded by the virus. The RDRP complex is required for viral mRNA expression and viral genome replication and is therefore essential for virus growth. Inhibition of the RDRP complex would arrest virus replication. The Basler and Shaw laboratories collaborated to optimize for 384-well high throughput screening a minigenome assay in which a functional EBOV RDRP complex is reconstituted by transfection of plasmids that express its four components into mammalian cells. RDRP activity is measured through the co-expression of a model viral RNA (minigenome RNA) that encodes a reporter gene flanked by the appropriate virus-derived cis-acting regulatory sequences. This system has been successfully transferred to collaborator Sumit Chanda at Sanford Burnham Prebys Medical Discovery Institute where a 6,400 compound pilot screen was performed. This screen yielded hits which were carried through to BSL4 testing by Robert Davey at Texas Biomedical Research Institute and demonstrated to inhibit Ebola virus replication. We propose to exploit this assay and this drug discovery pipeline to identify novel small molecule inhibitors of the Ebola virus polymerase. We will also develop additional HTS-compatible minigenome assays based on other filoviruses associated with deadly human disease, including Bundibugyo ebolavirus and Marburg virus, to identify and prioritize hits with pan-filovirus activity. A combination of minigenome assay and filovirus BSL4 experiments will define mechanisms of action, and together with initial SAR studies will prioritize hits for future development. The completion of these studies will significantly expand the number of potential therapeutic small molecules and provide significant insight into inhibition of the filovirus RDRP complex.